Priming Protein Pyrophosphorylation

Abstract

Inositol pyrophosphates are emerging players in the cell regulation arena. Although it was previously reported that diphosphoinositol pentakisphosphate [5-PP-Ins(1,2,3,4,6)P 5 , also abbreviated IP 7 ] phosphorylates proteins in yeast and mammals, Bhandari et al . now provide biochemical evidence that this phosphorylation is a pyrophosphorylation event with different chemical characteristics than phosphorylation mediated by ATP. The authors found that although yeast proteins were readily phosphorylated by IP 7 , when the same targets were expressed in bacteria, these proteins were not phosphorylated by IP 7 . The addition of yeast extract and ATP or GTP to the bacterially produced proteins restored IP 7 -mediated phosphorylation. Casein kinase 2 (CK2) is an enzyme that can use either GTP or ATP to phosphorylate target proteins. Phosphorylation of an IP 7 target expressed in bacteria (a fragment of protein NSR1) by CK2 or isolation of the NSR1 fragment from bacteria also expressing CK2 made the peptide a target for IP 7 -mediated phosphorylation, suggesting that there is a priming phosphorylation required for IP 7 -mediated phosphorylation. A synthetic peptide from another IP 7 and CK2 target, Nopp140, was phosphorylated by IP 7 , but only when the peptide was synthesized with the serine residues prephosphorylated. The number of sites phosphorylated by IP7 always correlated with the number of preexisting phosphates on the peptide, and methylation of phosphates prevented IP 7 -mediated phosphorylation of prephosphorylated peptides. A peptide in which the sites were pyrophosphorylated during synthesis was not a target for IP 7 -mediated phosphorylation. Together these data suggest that IP 7 mediates a pyrophosphorylation modification after priming by an enzymatic ATP-mediated phosphorylation event. Consistent with a different type of bond, the phosphorylation by IP 7 was more labile under acid conditions than was phosphorylation through an ATP-mediated process. In contrast, IP 7 -mediated phosphorylation was resistant to the action of phosphatases (λ-phosphatase, alkaline phosphatase, calcineurin, or protein phosphatase 1). Whether this type of pyrophosphorylation event influences protein activity and function and how this process might be regulated are interesting questions. R. Bhandari, A. Saiardi, Y. Ahmadibeni, A. M. Snowman, A. C. Resnick, T. Z. Kristiansen, H. Molina, A. Pandey, J. K. Werner Jr., K. R. Juluri, Y. Xu, G. D. Prestwich, K. Parang, S. H. Snyder, Protein pyrophosphorylation by inositol pyrophosphates is a posttranslational event. Proc. Natl. Acad. Sci. U.S.A. 104 , 15305-15310 (2007). [Abstract] [Full Text]